First tests
The main three tests that can be done after isolating a bacteria on media are the Oxidase test, the Catalase test, and Gram staining. These tests will show you the shape of the organism, its Gram stain, the presence/absence of Oxidase, and the organism's reactions to hydrogen peroxide. All organisms on this wiki are sorted by these qualities for the second set of tests. Plate technique The first step to any of these tests is to isolate the organism to be tested. There are several types of media used, but the technique in plating them is usually the same. The most popular method is quadrant streaking. This involves flaming an inoculation loop over a Bunsen burner until it glows red hot, letting it cool down, and then taking a sample of the organism from whatever it's primary media is. This sample is placed at the edge of an agar plate, and drug back and forth on its surface up to the 1/3 mark for the plate's diameter. The loop is flamed again and a sample drug out from the left side of the first quadrant (right side for left-handed individuals). The same pattern is repeated from that edge of the plate but take care to not overlap any of the quadrants. The loop is flamed again and the process repeated for a third quadrant. There should be a space left over that none of the previous quadrants touched. A single sample is drug from the 3rd quadrant into the edge of this and the sample is drug back and forth without overlapping for the final quadrant. After this the loop is sterilized a final time and the plate is ready to incubate. Gram staining This test requires an empty glass slide, a microscope, an inoculation loop, a Bunsen burner, crystal violet, Gram's iodine, ethanol, and safranin. A loop full of water (tap is fine) is added to the surface of the slide and a bit of organism is mixed into it and spread out over the slide. This is left to air dry until the water has evaporated. The slide is then passed over the flame of a Bunsen burner a few times to heat fix the bacteria. Crystal violet is applied to the slide and let sit for one minute. Excess dye is washed off with water before applying Gram's iodine solution for another minute. This is poured off but not rinsed with water, instead it is rinsed with ethanol until crystal violet has stopped leaking off of the slide. Lastly, safrinin is added to the slide for a final minute, before being rinsed off with tap water. This slide is now prepared, and can be observed with a microscope to see the shape and gram value of the organism. Microscopy In order to view the slide you've created you'll need a light microscope. Simply plug in and activate the light for the microscope and focus it in on low power. Switching to high enough power to see bacteria will often require you to add special oil to the slide first. This oil is often very dense and hard to remove from surfaces. Once the organism is fine focused, and often before, you'll be able to discern the dye that affixed itself to the cell wall. A blue color is indicative of Gram positive, and a red color is indicative of gram negative. There are some organisms that can be in-between, or resist reacting at all to this test, but none of them are in the list of organisms. Shape There are a multitude of possible shapes for bacteria, but the classic two shapes are bacillus (rods) and cocci. Cocci appear as roughly spherical shapes under the microscope while bacilli will appear elongated. Catalase test This test uses hydrogen peroxide as a reagent. The test can be performed in a number of ways, but the two most common methods either take a colony off of its growth media and mix it into a drop of hydrogen peroxide in a petri dish or other, similar container, or put the drop of hydrogen peroxide directly onto the colony on the media itself. If the organism is catalase positive, the peroxide will begin to foam almost immediately. It should be noted that hydrogen peroxide may start to foam anyway if left in contact with any organism for a long period of time, but this reaction is very slow. Only an immediate reaction is considered a positive result. Certain organisms create a very sticky substrate around themselves, and may not transfer properly to the peroxide. In these cases hold the transfer device in the drop for a while to check for reaction. Oxidase test This is testing for the presence of Cytochrome C Oxidase. This test either uses bottled reagent (which is transferred to paper or some absorbent media) or paper disks already soaked in this reagent. The organism is taken from the plate and smeared onto the reagent-soaked paper. After a 15 seconds, usually earlier, the part of the paper with the organism will begin to turn blue, indicating the oxidation of the reagent and the presence of Cytochrome C Oxidase. It is important to note this test works the best on fresh plates. Do not use inoculating loops to transfer bacteria, the materials they're typically made with can cause false positives. Starting the Key Once all of these tests have been completed the basis for the key is complete and it can be started here.